This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in amino acid biosynthesis in plants and seeds.
Many vertebrates, including man, lack the ability to manufacture a number of amino acids and therefore require these amino acids preformed in the diet. These are called essential amino acids. Human food and animal feed, derived from many grains, are deficient in essential amino acids, such as lysine, the sulfur amino acids methionine and cysteine, threonine and tryptophan. For example, in corn (Zea mays L.) lysine is the most limiting amino acid for the dietary requirements of many animals. Soybean (Glycine max L.) meal is used as an additive to com-based animal feeds primarily as a lysine supplement. Thus, an increase in the lysine content of either corn or soybean would reduce or eliminate the need to supplement mixed grain feeds with lysine produced via fermentation of microbes. Furthermore, in corn the sulfur amino acids are the third most limiting amino acids, after lysine and tryptophan, for the dietary requirements of many animals. The use of soybean meal, which is rich in lysine and tryptophan, to supplement corn in animal feed is limited by the low sulfur amino acid content of the legume. Thus. an increase in the sulfur amino acid content of either corn or soybean would improve the nutritional quality of the mixtures and reduce the need for further supplementation through addition of more expensive methionine.
Lysine, threonine, methionine, cysteine and isoleucine are amino acids derived from aspartate. Regulation of the biosynthesis of each member of this family is interconnected (see FIG. 1). One approach to increasing the nutritional quality of human foods and animal feed is to increase the production and accumulation of specific free amino acids via genetic engineering of this biosynthetic pathway. Alteration of the activity of enzymes in this pathway could lead to altered levels of lysine, threonine, methionine, cysteine and isoleucine. However, few of the genes encoding enzymes that regulate this pathway in plants, especially corn, soybeans and wheat, are available.
The organization of the pathway leading to biosynthesis of lysine, threonine, methionine, cysteine and isoleucine indicates that over-expression or reduction of expression of genes encoding, inter alia, threonine synthase, dihydrodipicolinate reductase, diaminopimelate epimerase, threonine deaminase and S-adenosylmethionine synthetase in corn, soybean, wheat and other crop plants could be used to alter levels of these amino acids in human food and animal feed. Accordingly, availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate development of nutritionally improved crop plants.
The instant invention relates to isolated nucleic acid fragments encoding plant enzymes involved in amino acid biosynthesis. Specifically, this invention concerns isolated nucleic acid fragments encoding the following plant enzymes that catalyze steps in the biosynthesis of lysine, threonine, methionine, cysteine and isoleucine from aspartate:
dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-adenosylmethionine synthetase. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding the listed plant biosynthetic enzymes.
In another embodiment, the instant invention relates to chimeric genes encoding the amino acid biosynthetic acid enzymes listed above or to chimeric genes that comprise nucleic acid fragments that are complementary to the nucleic acid fragments encoding the enzymes, operably linked to suitable regulatory sequences. wherein expression of the chimeric genes results in production of levels of the encoded enzvmes in transformed host cells that are altered (i.e., increased or decreased) from the levels produced in untransformed host cells.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a plant amino acid biosynthetic enzyme operably linked to suitable regulatory sequences, the enzyme selected from the group consisting of: dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-adenosylmethionine synthetase. Expression of the chimeric gene results in production of altered levels of the biosynthetic enzyme in the transformed host cell. The transformed host cells can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a plant biosynthetic enzyme in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a plant biosynthetic enzyme selected from the group consisting of dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-adenosylmethionine synthetase, operably linked to suitable regulatory sequences; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the biosynthetic enzyme in the transformed host cell.
An additional embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or substantially all of an amino acid sequence encoding a plant dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-adenosylmethionine synthetase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a plant biosynthetic enzyme selected from the group consisting of dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-adenosylmethionine synthetase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a plant biosynthetic enzyme selected from the group consisting of dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-denosylmethionine synthetase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the biosynthetic enzyme in the transformed host cell; (c) optionally purifying the biosynthetic enzyme expressed by the transformed host cell; (d) treating the biosynthetic enzyme with a compound to be tested; and (e) comparing the activity of the biosynthetic enzyme that has been treated with a test compound to the activity of an untreated biosynthetic enzyme, thereby selecting compounds with potential for inhibitory activity.